There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, since there is no methodology to turn particles ‘off.’ We have developed a simple technique to rapidly remove silver nanoparticles outside living cells leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate/thiosulfate redox-based destain solution. In demonstration of the technique we present a new class of multicolored plasmonic nanoprobes comprising dye-labeled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice and that show tumour uptake in vivo.

Functional inactivation of p53 and constitutive activation of the NF-B pathway has been associated with several human cancers. In this study, we show that I B kinase 2 (IKK2/IKK␤), which is critical for NF-B activation, also phosphorylates p53. Phosphorylation of p53 at serines 362 and 366 by IKK2 leads to its recruitment to and ubiquitination by ␤-TrCP1. Degradation of ubiquitinated p53 is independent of Mdm2, because it occurs in both wild-type and Mdm2 ؊/؊ cells. SiRNA-mediated reduction in the levels of ␤-TrCP1 and other members of the SCF ␤؊TrCP1 E3 ubiquitin ligase complex or overexpression of a dominant negative form of ␤-TrCP1 enhances p53 stability. Substitutions at Ser-362 and 366 of p53 by alanines (p53 AA) result in reduced phosphorylation of p53 by IKK2, decreased association with ␤-TrCP1, and thus increased stability of p53 and expression of p53 target genes such as p21, altering the G1 phase of the cell cycle. Our results identify IKK2 and ␤-TrCP1 as novel regulators of the p53 pathway and suggest that blocking of IKK2 and ␤-TrCP1 could be a means of regulating p53 stability and thereby modulating its biological activity.␤-TrCP1 ͉ DNA damage ͉ IKK2 ͉ p53 stability N F-B is a family of transcription factors crucial for several biological processes involved in innate and adaptive immunity, inflammation, cell survival, and cancer (1). More than 150 different types of stimuli, including proinflammatory cytokines, bacterial/ viral infection, and stress, activate NF-B-dependent transcription of target genes. Activation of the I B kinase (IKK) complex is a prerequisite for NF-B activation. Once activated, IKK phosphorylates serines 32 and 36 of I B proteins (2), followed by ubiquitination by ␤-TrCP1, an E3 ubiquitin ligase, which targets them for proteosomal degradation (3). IKK2 has been shown to phosphorylate other cellular and viral proteins, such as ␤-catenin, I B␤, I B , p65, HIV-VPU, and SRC-3; this phosphorylation regulates the steady-state levels and transcriptional activity of these substrates (4-7). Approximately 30% of cellular proteins contain covalently bound phosphate, even though only 500-600 protein kinases likely are encoded by the human genome (8). Therefore, it is conceivable that, like many other kinases, IKK2 also has additional substrates, and that each protein phosphorylated by IKK2 in turn can be a substrate of other kinases.The p53 tumor suppressor plays a crucial role in the development of many cancers and is frequently mutated or deleted in human cancers (9, 10). In the absence of stress, endogenous p53 levels are very low, because of the constant recruitment of p53 to Mdm2, an E3 ubiquitin ligase that inhibits its transcriptional activity and targets it for proteosomal degradation (11,12). In response to DNA damage and other cellular stresses, the p53 protein levels are up-regulated and its activities are induced. The inhibition of Mdm2 binding to p53 and increases in p53 transcriptional activity involve the phosphorylation and stabilization of p53.Human p53 has been reported ...

Tumor-specific tissue-penetrating peptides deliver drugs into extravascular tumor tissue by increasing tumor vascular permeability through interaction with neuropilin (NRP). Here we report that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice. The anti-metastatic effect was mediated by the NRP-binding RXXK peptide motif (CendR motif), and not by the integrin-binding RGD motif. iRGD inhibited migration of tumor cells and caused chemorepulsion in vitro in a CendR- and NRP-1-dependent manner. The peptide induced dramatic collapse of cellular processes and partial cell detachment, resulting in the repellent activity. These effects were prominently displayed when the cells were seeded on fibronectin, suggesting a role of CendR in functional regulation of integrins. The anti-metastatic activity of iRGD may provide a significant additional benefit when this peptide is used for drug delivery to tumors.

Peritoneal carcinomatosis is a major source of morbidity and mortality in patients with advanced abdominal neoplasms. Intraperitoneal chemotherapy (IPC) is an area of intense interest given its efficacy in ovarian cancer. However, IPC suffers from poor drug penetration into peritoneal tumors. As such, extensive cytoreductive surgery is required prior to IPC. Here, we explore the utility of iRGD, a tumor-penetrating peptide, for improved tumor-specific penetration of intraperitoneal compounds and enhanced IPC in mice. Intraperitoneally administered iRGD significantly enhanced penetration of an attached fluorescein into disseminated peritoneal tumor nodules. The penetration was tumor-specific, circulation-independent, and mediated by the neuropilin-binding RXXK tissue-penetration peptide motif of iRGD. Q-iRGD, which fluoresces upon cleavage, including the one that leads to RXXK activation, specifically labeled peritoneal metastases displaying different growth patterns in mice. Importantly, iRGD enhanced intratumoral entry of intraperitoneally co-injected dextran to approximately 300% and doxorubicin to 250%. Intraperitoneal iRGD/doxorubicin combination therapy inhibited the growth of bulky peritoneal tumors and reduced systemic drug toxicity. iRGD delivered attached fluorescein and co-applied nanoparticles deep into fresh human peritoneal metastasis explants. These results indicate that intraperitoneal iRGD co-administration serves as a simple and effective strategy to facilitate tumor detection and improve the therapeutic index of IPC for peritoneal carcinomatosis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked desmoplasia and drug resistance due, in part, to poor drug delivery to extravascular tumor tissue. Here, we report that carcinoma-associated fibroblasts (CAFs) induce β5 integrin expression in tumor cells in a TGF-β dependent manner, making them an efficient drug delivery target for the tumor-penetrating peptide iRGD. The capacity of iRGD to deliver conjugated and co-injected payloads is markedly suppressed when β5 integrins are knocked out in the tumor cells. Of note, β5 integrin knock-out in tumor cells leads to reduced disease burden and prolonged survival of the mice, demonstrating its contribution to PDAC progression. iRGD significantly potentiates co-injected chemotherapy in KPC mice with high β5 integrin expression and may be a powerful strategy to target an aggressive PDAC subpopulation.

Lifeguard (LFG) is an inhibitor of Fas-mediated cell death and is highly expressed in the cerebellum. We investigated the biological role of LFG in the cerebellum in vivo, using mice with reduced LFG expression generated by shRNA lentiviral transgenesis (shLFG mice) as well as LFG null mice. We found that LFG plays a role in cerebellar development by affecting cerebellar size, internal granular layer (IGL) thickness, and Purkinje cell (PC) development. All these features are more severe in early developmental stages and show substantial recovery overtime, providing a remarkable example of cerebellar plasticity. In adult mice, LFG plays a role in PC maintenance shown by reduced cellular density and abnormal morphology with increased active caspase 8 and caspase 3 immunostaining in shLFG and knockout (KO) PCs. We studied the mechanism of action of LFG as an inhibitor of the Fas pathway and provided evidence of the neuroprotective role of LFG in cerebellar granule neurons (CGNs) and PCs in an organotypic cerebellar culture system. Biochemical analysis of the Fas pathway revealed that LFG inhibits Fas-mediated cell death by interfering with caspase 8 activation. This result is supported by the increased number of active caspase 8-positive PCs in adult mice lacking LFG. These data demonstrate that LFG is required for proper development and survival of granular and Purkinje cells and suggest LFG may play a role in cerebellar disorders.

In the secondary lymphoid organs, intimate contact with follicular dendritic cells (FDCs) is required for B cell retention and antigen-driven selection during the germinal center response. However, selection of self-reactive B cells by antigen on FDCs has not been addressed. To this end, we generated a mouse model to conditionally express a membrane-bound self-antigen on FDCs, and monitor the fate of developing self-reactive B cells. Here, we show that self-antigen displayed on FDCs mediates effective elimination of self-reactive B cells at the transitional stage. Notwithstanding, some self-reactive B cells persist beyond this checkpoint, showing evidence of antigen experience and intact proximal BCR signaling, but they are short-lived and unable to elicit T cell help. These results implicate FDCs as an important component of peripheral B cell tolerance that prevent the emergence of naïve B cells capable of responding to sequestered self-antigens.

Organotypic tissue slices prepared from patient tumors are a semi-intact ex vivo preparation that recapitulates many aspects of the tumor microenvironment (TME). While connections to the vasculature and nervous system are severed, the integral functional elements of the tumor remain intact for many days during the slice culture. During this window of time, the slice platforms offer a suite of molecular, biomechanical and functional tools to investigate PDAC biology. In this review, we first briefly discuss the development of pancreatic tissue slices as a model system. Next, we touch upon using slices as an orthogonal approach to study the TME as compared to other established 3D models, such as organoids. Distinct from most other models, the pancreatic slices contain autologous immune and other stromal cells. Taking advantage of the existing immune cells within the slices, we will discuss the breakthrough studies which investigate the immune compartment in the pancreas slices. These studies will provide an important framework for future investigations seeking to exploit or reprogram the TME for cancer therapy.